Chlorophyll, one of the major chloroplast components for photosynthesis, has a positive relationship with the photosynthetic rate. The chlorophyll content is an important assessment parameter in agronomy and plant biology research. This study was conducted to evaluate the natural variation in the chlorophyll content and to determine the differential response of the chlorophyll concentration to dark treatment in a natural population containing 139 maize inbreds. A five-fold higher chlorophyll concentration was measured in the light compared with the dark. Meanwhile, the wide variation in the chlorophyll concentration showed the differential response of the natural maize population to dark. Finally, we identified some inbreds that were highly sensitive to the dark with more than 70% difference between the light and dark treatment, such as Dan598, Zheng29, Zheng35, DH29, and R08, as well as some inbreds that had lower sensitivity to the dark, with less than 35% difference in the chlorophyll content between the light and dark treatment, such as Chuan48-2, 4F1, 303WX, 9642, and LY042.
Maize Inbred Lines Seedlings Chlorophyll Dark Treatment1. Introduction
As the most important pigment in the world, chlorophyll (Chl) plays a central role in photosynthesis and is responsible for absorbing sunlight and converting it into chemical energy [1] . Maintaining a higher Chl content for a longer period in the reproductive stage is essential for increasing crop production [2] [3] . However, excess Chl in its free state produces reactive oxygen species and results in cell death [4] ; therefore, plant cells must degrade these species through self-metabolism. Green plants contain two major Chl components, chlorophyll a (Chla) and chlorophyll b (Chlb), both with the same absorption spectra [5] [6] . Chla and Chlb function as photoreceptors in photosynthesis. Chla is a unique pigment that exists in all oxygenic photosynthetic organisms [7] . Chlb functions in the light-harvesting Chla/b protein complexes (LHCs) that harvest and transfer light energy to both photosystems [8] .
The Chl content is an important experimental parameter in agronomy and plant biology research [9] . The Chl content is used as an effective index to assess photosynthetic efficiency in breeding programs [10] . This parameter is fundamental to understanding the response of a plant to the adverse environment in which it resides. Because it is a quantitative trait, it is difficult to select for Chl content in breeding programs [11] . The amount of Chl varies depending on many edaphic and climatic factors, such as salt stress [12] -[14] , light [15] -[19] , water stress [20] -[25] , air pollution [26] , fertilization [27] , the vegetation period [28] , plant species and leaf position [29] . Therefore, the tolerance of plants for stresses such as cold and drought [30] -[33] or low temperature [34] [35] can be determined by the Chl concentration.
Maize (Zea mays L.) is the world’s most widely grown crop. It is also an important source of biofuel, animal feed and raw material in industry [36] . In addition, maize is an important model organism for cytogenetic, genetic, genomic, and functional genomic studies based on complete whole genome sequencing [37] . Increasing the grain yield and biomass per acre is one of the most important goals of maize production [38] . Nowadays, adverse weather and environmental diversity threaten maize yield, which has resulted in a research hotpot on the maize response to adverse environmental conditions. In this present study, a natural population with 139 inbred maize lines was used to investigate the variation in the Chl content before and after dark treatment and to evaluate the response of different inbred lines to dark treatment.
2. Materials and Methods2.1. Experimental Conditions and Treatments
The inbred maize lines were provided by the National Maize Improvement Centre of China [39] . Two seed replicates were germinated in petri dishes for 4 days and transplanted to enriched soil (light nutritional soil: vermiculite = 1:1) under the following growth conditions: light/dark: 10/14 hours; temperature: 25˚C; white light: 400 μmol∙m−2∙s−1. When the second leaf had emerged completely, one replicate was placed under light and the other was placed in the dark for four days. All the phenotypic data were collected after four days of dark treatment.
2.2. Phenotypic Determination and Analysis
When two leaves had fully unfolding, two types of methods were used for Chl determination before and after 4 days of dark treatment. The first method was the use of the SPAD-502 chlorophyll meter (Minolta Camera Co., Osaka, Japan) and the second was the chemical method in which Chl from the green part of three plants was extracted using 80% acetone and measured using spectrophotometer at wavelengths of 645 nm and 663 nm [40] . All data were analyzed by using SPSS v.19 software and the Microsoft Excel program, including basic statistics description, mono factor analysis of variance and Pearsoncorrelation. The relative contents of chlorophyll a, b and total chlorophyll (Tchl = Chla + Chlb) and Chla/b (Chla/Chlb) were calculated using the following equations [40] .
where A645 = absorbance at 645 nm; A663 = absorbance at 663 nm; V = solvent volume, W = fresh weight of the extracted tissue, N = dilution factor.
3. Results3.1. Phenotypic Analysis
In general, there was wide variation in the SPAD value and the Chl concentration measured in the light and dark. The average SPAD values were 38.02 and 29.83 under light and dark conditions, respectively, and the variation was double that measured in the natural population. The largest values were 48.35 from ZaC546 and 42.12 from Shen137, and the smallest SPAD values were 26.33 from NMJT and 18.32 from HSBN under light and dark conditions, respectively. This suggested that a smaller variation of 22.03 was observed in the light compared with a greater variation of 23.80 in the dark (Table 1).
For the Chla, Chlb, and Tchl concentrations, there was two-fold difference between the light and dark for the average values; however, a small difference was observed in Chla/b under light and dark conditions. The average Chla concentration was 0.57 mg/g (range = 1.03 mg/g) in the light, compared with 0.22 mg/g (range = 0.54 mg/g) in the dark. The variation of Chlain the population was more than ten-fold, and ranged from 0.18 - 1.21 mg/g and 0.04 - 0.58 mg/g under light and dark conditions, respectively. The highest concentration was measured in Mo17 and the lowest in K14 in the light, compared with the highest concentration in TY-2 and the lowest in Zheng-35 in the dark. The Chlb concentrations ranged from 0.26 mg/g (238) to 0.96 mg/g (Qi319) in the light, i.e., almost a four-fold variation, compared with the range from 0.06 mg/g (Dan598) to 0.58 mg/g (TY-2) in the dark, i.e., almost a ten-fold variation. The average Chlb concentrations determined in the light and dark were 0.52 mg/g and 0.26 mg/g, respectively. The Tchl concentration in the light ranged from 0.55 - 2.05 mg/g, with a high variation of 1.51 mg/g and an average value of 1.09 mg/g, compared with the range of 0.12 - 1.16 mg/g, a variation of 1.05 mg/g and an average of 0.49 mg/g measured under dark conditions. The Chla/b range was 0.44 - 1.49 and the average was 1.07 under light conditions, compared with the range of 0.09 - 1.21 and the average of 0.84 under dark conditions. When we measured the Chla and Chlb percent in Tchl under light and dark conditions, only a small difference was found between the average values. A two-fold variation in the Chla percent in Tchl (range of 30.95 - 59.92) was observed in the light, and a seven-fold variation was measured in the dark (range of 8.26 - 54.79). With respect to the Chlb percent in Tchl, there was a two-fold variation under the light and dark conditions, where ranges of 40.08 - 69.05 and 45.21 - 91.74, respectively, were measured (Table 1). Under light conditions, a higher Chla percent was observed compared with a lower value under dark conditions. This indicates that the seedlings required the condition of a distribution in the Chla and Chlb concentrations to adapt to changes in the environment.
3.2. Correlation Coefficients between the SPAD Readings and the Chl Concentration
To explore the relationship between the SPAD readings and the Chl concentration, Pearson correlation coefficients were calculated for the different treatments as shown in Table 2. For the SPAD readings, there was a highly significant relationship, with a value of 0.561, between the light and dark. However, no significant correlation was detected between the SPAD readings and the Chl concentration for the light or dark treatment. Furthermore, there was no significant correlation between the light and dark treatment for a particular Chl component, including Chla/b. Chla under light conditions was highly significantly positively correlated with Chlb (0.845), Tchl (0.973), and Chla/b (0.608), similar to the dark conditions. In addition, a highly significant positive relationship was also found between Chlb and Tchl, i.e., 0.944 and 0.949 under light and dark conditions, respectively. Chla/b had a highly significant positive correlation with Chla (0.608) but not with Chlb or Tchl under light conditions, and similar associations were observed in the dark. There was no significant difference in the chlorophyll components between the different treatments (Table 2).
3.3. Difference in Chl Concentration under Light and Dark Conditions
The relative difference in the Chl content between the light and dark treatments was also analyzed for this population, as shown in Table S1. It was found that the Top 10 least difference for Tchl was less than 35%, and the inbreds were Chuan48-2, 4F1, 303WX, 9642, and LY042. However, the difference between the Chla and Chlb contents was less than 40%. Therefore, we considered that these inbreds could be insensitive to dark treatment. On the other hand, it was determined that the inbreds Dan598, Zheng29, Zheng35, DH29, and R08 obtained the Top highest difference percent of more than 70% for Tchl and Chla, and a difference percent of more than 60% for Chlb. Compared with Chlb, Tchl and Chla showed a more rapid degradation under dark treatment, which suggested that these inbreds might be highly sensitive to dark treatment.
In particular, there was a miniscule increment in Chla in inbreds 4F1 and 9642, with a small decrease in Tchl and a higher decrease in Chlb. Meanwhile, there was a small increment in Chla in inbred 303WX, which exhibited a 15% decrease in Chla and a 7% decrease in Tchl. This result verified that the mode of transition between Chla and Chlb during the dark treatment differed between individual inbred lines (Table S1).
4. Discussion
A significant relationship between SPAD readings and the Chl concentration was determined by using chemical extraction in a previous report [41] ; however, this relationship was not significant in the current study. It was possible that a different leaf part was used for the chemical extraction. In this study, which was in contrast to previous reports, only the middle section of the first leaf was measured by using the SPAD-502 meter, and all the green parts of the seedling were used for Chl determination. In other studies, however, the same part of the ear leaf was used to evaluate the Chl content.
According to differences in their Chl contents between light and dark, inbred lines were identified as sensitive or insensitive to dark. Inbreds such as Dan598, Zheng29, Zheng35, DH29, and R08 will be likely senescence rapidly under dark conditions, and Chuan48-2, 4F1, 303WX, 9642, and LY042 exhibit a stay-green phenotype with slow senescence in the dark. In addition, there were several inbred lines that exhibited a miniscule increment in one type of Chl but a decrease in another type of Chl and in Tchl. It can be concluded that the mechanism of transition between Chla and Chlb is different in different inbred lines under dark conditions.
Contrary to our expectations, Qi319 was not identified as a stay-green inbred under dark induced-senescence at the seedling stage, although it was classified as a stay-green type at the mature stage according to reference [41] . We infer that the mechanisms in the maize leaf differ under natural and induced senescence. This is consistent with the conclusion from the differential expression analysis using transcription analysis [42] .
5. Conclusion
The amount of Chl in leaves varies and is affected by many factors. In this study, it was observed that the Chl concentration was five-fold higher in the light than in the dark, and inbreds that were sensitive or insensitive to dark treatment were identified according to their different response to dark. During the dark treatment, the transition between Chla and Chlb was different in the different inbreds. Moreover, a difference was observed between natural and induced leaf senescence in maize. Determination of Chl content can be used in many field studies, and its related research should be increased and performed for efficient use of Chl content.
Acknowledgements
We greatly acknowledge Dr. Yan J.B. from Huazhong Agriculture University and Dr. Yang X.H. from China Agriculture University for supplying the inbred maize lines. We also thank for the foundation of Chinese Universities Scientific Fund of Northwest A&F University (Z109021515) and the PhD research startup foundation of Northwest A&F University (Z109021408). We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
Author Contributions
Conceived and designed the experiments: Xue, J. Q. and Xu, S. T.; Performed the experiments: Aye, N. C., Shi, Y. Q. and Li, Y. N.; Analyzed the data: Aye, N. C. and Xu, S. T.; Contributed to modify the manuscripts: Li, Y. J., Guo, D. W. and Xue, J. Q.; Wrote the paper: Xu, S. T. and Aye, N. C.,
Competing Interests
The authors have declared that no competing interests exist.
Cite this paper
Aye NyeinChan,11,ShutuXu,11,DongweiGuo,11,YaqinShi,11,YananLi,11,YajunLi,11,JiquanXue,11, (2015) Dark Response of Seedlings Evaluated by Chlorophyll Concentration in Maize Natural Population. American Journal of Plant Sciences,06,2209-2219. doi: 10.4236/ajps.2015.613223
Appendices
Descriptive statistics of traits for Chl content in inbred lines population
Trait
SPAD-L
SPAD-D
Chla-L
Chla-D
Chlb-L
Chla-D
TChl-L
TChl-D
Chla/b-L
Chla/b-D
Chla%-L
Chla%-D
Chlb%-L
Chlb%-D
Max
48.35
42.12
1.21
0.58
0.96
0.58
2.05
1.16
1.49
1.21
59.92
54.79
69.05
91.74
Min
26.33
18.32
0.18
0.04
0.26
0.06
0.55
0.12
0.44
0.09
30.95
8.26
40.08
45.21
Range
22.02
23.80
1.03
0.54
0.71
0.52
1.51
1.05
1.05
1.12
28.97
46.53
28.97
46.53
Mean
38.02
29.83
0.57
0.22
0.52
0.26
1.09
0.49
1.07
0.84
51.33
45.13
48.66
54.87
SD
4.34
4.81
0.18
0.10
0.13
0.10
0.30
0.19
0.19
0.17
4.85
5.85
4.85
5.85
Note: SPAD value, Chlorophyll compounds content; Chla, Chlb: chlorophyll a and chlorophyll b content (mg/g); TChl: total chlorophyll (Chla + b) (mg/g); Chla/b: the ratio of chlorophyll a and b content; Chla%, Chlb%: chlorophyll a and b percent in total chlorophyll content; SD = standard deviation, SPAD reading and Chla/b have no unit.
Correlation coefficients for SPAD readings and Chl concentration
Trait
SPAD-L
SPAD-D
Chla-L
Chlb-L
TChl-L
Chla/b-L
Chla-D
Chlb-D
TChl-D
Chla/b-D
SPAD-L
1
SPAD-D
0.561**
1
Chla-L
0.165
0.181
1
Chlb-L
0.280
0.257
0.845**
1
TChl-L
0.215
0.213
0.973**
0.944**
1
Chla/b-L
−0.091
−0.050
0.608**
0.229
0.437
1
Chla-D
0.068
0.186
0.245
0.295
0.265
0.068
1
Chlb-D
0.075
0.207
0.274
0.373
0.319
−0.024
0.722**
1
TChl-D
0.051
0.177
0.267
0.339
0.297
0.029
0.951**
0.949**
1
Chla/b-D
−0.144
0.016
0.153
0.014
0.088
0.350
0.533**
0.258
0.436
1
Note: SPAD: SPAD readings; Chla: Chlorophyll a; Chlb: Chlorophyll b; TChl: total chlorophyll (Chl a + b); Chla/b: the ratio of chlorophyll a and b; L: light; D: dark; **Significant at the 0.01 probability level.
Supplementary Information
Relative chlorophyll differences between selected inbreds under dark treatment
Inbred Lines
Differential %
Chla
Chlb
Tchl
chuan48-2
2
5
3
4F1
−6
14
5
303WX
15
−1
7
9642
−2
21
10
LY042
−32
34
12
B77
35
14
24
526018
30
22
26
TY2
33
18
26
4019
34
19
27
04K5686
33
22
28
384-2
31
31
31
05W002
31
32
32
238
39
26
33
M97
31
36
34
B113
36
32
34
EN25
46
22
35
D863F
43
26
35
LY
41
33
36
JY01
21
46
36
MN
39
32
36
B73
49
21
36
B110
36
37
36
C8605
49
26
37
L3180
37
38
38
Zheng30
37
39
38
Dan360
36
40
38
A619
46
29
38
FCD0602
42
37
39
835a
36
43
40
3411
41
38
40
Si273
49
34
41
Chang3
43
40
42
Lx9801
42
42
42
18-599
46
38
42
K12
45
42
43
LG001
45
42
43
04K5702
42
46
44
835b
47
41
44
TY1
52
35
44
501
47
42
45
C17
59
27
45
U8112
53
36
45
TY4
49
43
46
K14
30
54
47
Qi205
52
40
47
TY3
50
43
47
Zi330
53
40
47
07KS4
59
54
56
Shen137
64
48
56
Shen5003
64
48
57
Zheng22
63
50
57
268
61
54
57
TY9
63
52
58
7884-4Ht
66
50
59
Zheng28
66
49
59
Tian77
64
51
59
05WN230
58
60
59
Ye8001
64
52
59
Liao159
65
51
59
5213
64
54
59
TT16
65
55
59
HSBN
68
49
59
JH96C
65
53
60
IRF314
57
62
60
Ji63
64
57
60
HTH-17
66
56
61
Jiao51
65
55
61
975-12
72
52
62
Si434
70
52
62
Tie7922
70
54
63
Yan414
70
56
63
Q1261
71
56
63
Xun971
77
51
64
1323
64
64
64
R15X1141
68
60
64
Dan4245
70
56
64
TX5
67
62
64
K10
68
62
65
Dan3130
67
63
65
Ye52106
73
56
65
Yu87-1
70
60
65
7327
72
58
65
Dan340
81
45
65
Ye515
70
62
67
Mo17
71
61
67
8902
69
64
67
Liao138
74
60
67
Chang7-2
75
57
67
Ye478
73
63
68
W138
69
67
68
Lv28
71
65
68
J4112
76
61
69
ZB648
72
65
69
1462
51
46
48
WH413
44
53
49
812
51
49
50
Yu374
57
43
51
LXN
60
42
51
M153
56
47
52
5311
59
45
52
Dan599
58
47
52
M165
55
50
53
B111
61
46
53
HB
61
44
53
TY10
60
47
53
Zhi41
62
46
54
B151
60
47
54
HYS
59
50
54
Ji53
62
43
54
S22
61
46
55
P178
61
50
55
Zheng58
94
4
56
K22
60
51
56
R15
73
71
72
P138
77
66
72
Liao5114
78
66
72
ES40
75
69
72
Dan9046
78
65
73
Ji846
77
69
73
Si444
75
70
73
Zheng32
77
69
74
3H-2
76
72
74
HuangC
80
69
74
Zong3
78
70
75
Qi319
77
73
75
NMJT
82
71
78
ZaC546
82
74
78
R08
86
70
78
DH29
85
72
79
Zheng35
92
80
86
Zheng29
93
83
88
Dan598
92
88
90
JH59
48
46
47
Ji853
50
45
47
Si446
84
48
69
TY7
76
64
71
7381
49
44
47
Dong46
75
63
69
Nan21-3
74
63
69
Note: Chla: Chlorophyll a; Chlb: Chlorophyll b; Tchl: Total chlorophyll (Chl a + b).
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